Research addressing the main specific aims of this project focused on cellular and circuit mechanisms generating the respiratory rhythm and neural activity patterns in the brainstem of rodents. Experimental studies were performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and mature rats and mice. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further exploited methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing structural imaging performed simultaneously with functional activity imaging by multi-photon laser scanning microscopy of the neurons labeled with fluorescent calcium-sensitive dyes and/or fluorescent proteins. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron channels, receptors, and neurotransmitter-related proteins. With these approaches, we have performed high-resolution spatiotemporal imaging of neuron activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro. These studies have provided the most direct experimental evidence to date that rhythm generation involves an excitatory network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. By applying optogenetic approaches we have confirmed that a critical population of glutamatergic neurons with voltage-dependent oscillatory properties are the substrate for inspiratory rhythm generation in the pre-Botzinger complex. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with intracellular recording techniques in situ and advanced electrophysiolgical approaches such as the dynamic clamp applied in vitro, continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratory rhythm and pattern in the intact mammalian nervous system. Studies in progress based on intracellular recording approaches applied in situ are analyzing in detail how distinct populations of excitatory and inhibitory neurons interact to generate the respiratory rhythm and pattern as well as to test predictions of our network models. Furthermore, optogenetics-based studies involving photo-inhibition or photo-excitation of inhibitory respiratory neurons has established a fundamental role of inhibitory microcircuits including in the pre-Botzinger complex in respiratory pattern generation. Other studies have provided additional evidence that neuronal persistent sodium currents and several types of leak or background conductances represent critical ionic conductance mechanisms for generation and control of respiratory oscillations. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons in vitro, as well as our current immunohistochemical and pharmacological studies, have identified a specialized set of transient receptor potential (TRP) cationic channels that also represent important regulators of neuron excitability and current studies are directed toward understanding how these channels may contribute to electrophysiological behavior of respiratory circuit neurons. Other electrophysiological studies have demonstrated that leak conductance mechanisms are critically involved in the regulation of rhythmic breathing patterns by a diverse set of endogenous neurochemicals that modulate these conductances as well as by physiological control signals including carbon dioxide and oxygen. A particular focus of these latter studies was elucidating neuromodulatory control of respiratory circuit activity by neurons of the brainstem raphe nucleus that constitute the brainstem serotonin (5-HT) system, which is postulated to have a critical function in brain state-dependent control of breathing in vivo and is associated with pathophysiological disturbances of breathing such as thosse underlying sudden infant death syndrome (SIDS). Our continuing electrophysiological studies performed in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and have determined the essential modulatory actions of raphe 5-HT neurons in both the neonatal and mature mammalian nervous systems. Previously we have shown that raphe 5-HT neurons have slow pacemaking properties dependent in part on the kinetic properties of sodium and leak channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. We have also continued to analyze how pharmacological activation of various types of 5-HT receptors, especially 5-HT(1a) receptors, on different populations of respiratory circuit neurons affects circuit activity and how this can be exploited to reverse opioid-induced depression of breathing with potential translational therapeutic applications. In previous studies employing novel pharmacogenetic approaches applied in situ and in vivo, neurons of the retrotrapezoid nucleus (RTN) that also have slow pacemaking and chemosensory properties were also shown to provide a critical excitatory modulatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. Accordingly new models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory input control mechanisms have been formulated to explain how specific brainstem circuit components are controlled and regulate patterns of respiratory oscillatory activity. We are currently extending the optogenetics-based studies to manipulate activity of specific neuronal populations to further investigate how different populations of network neurons contribute to respiratory pattern generation in various (patho)physiological states.